PIWI/piRNA在精子细胞中的翻译激活作用研究

A unique feature in late stages of male-germ cell development is the remarkable compaction of sperm chromatin, which results in a drastic inhibition of transcriptional activity at spermiogenesis, the late stages of spermatogenesis. In this regard, the genes that are required for late spermatids and spermatozoa need to be transcribed in advance and stored as mRNAs in earlier stages of germ cells, including spermatocytes and round spermatids. This event states the critical importance of post-transcriptional regulation in spermiogenesis; however, the mechanisms underlying have remained largely unexplored. piRNAs are a novel class of animal germ cell-specific small noncoding RNAs that interact to PIWI family proteins. Recent studies indicate that PIWI/piRNA prevents the activity of mobile genetic elements from destabilizing DNA in animal germ cells, which protects the integrity of germ cell genome and thus is essential to gametogenesis. Our preliminary results showed that a few spermatid-specific mRNAs were co-immunoprecipitated with MIWI protein through RNA immunoprecipitation and deep sequencing assays. Intriguingly, our reporter assays showed that MIWI, in complex with specific piRNAs, up-regulated the expression of these mRNAs. These results suggest that PIWI/piRNAs, besides silencing mobile genetic elements in genome, are also involved in the regulation of gene expression in spermatids. Consistently, we found that MIWI interacted to eIF3f, a subunit of translational initiation factor eIF3 complex. In this research plan, we will study the physiological role and mechanism of MIWI/piRNA-mediated translational activation in spermiogenesis, and explore the molecular mechanism of post-transcriptional regulation of gene expression in late spermatids and spermatozoa. The obtained results may bring new insights into molecular information of abnormal development of spermatids and dysregulation of sperm maturation, and also may provide molecular target for diagnosis and therapeutics of male infertility.

在精子发生后期，因染色质被高度压缩致转录停止，为后期发育所需基因需提前转录为mRNA储存，因而转录后水平调控对精子形成和成熟至关重要，但目前对相关调控的分子机制还知之甚少。piRNA是一类动物生殖细胞特异性小分子非编码RNA。研究发现,piRNA特异性地与PIWI蛋白结合，沉默基因组移动遗传元件，维持生殖细胞基因组稳定性和完整性，为配子形成所必需。我们最近的研究发现，小鼠PIWI蛋白MIWI/piRNA结合多个精子细胞特异性mRNA并促进其表达，提示PIWI/piRNA还具有调控精子细胞基因表达的新功能；与之一致的是，我们还发现MIWI与翻译起始因子eIF3复合物亚基eIF3f相互作用。本项目将调查MIWI/piRNA介导的翻译激活作用在精子形成和成熟中的功能，解析精子细胞中转录后水平调控的分子机制。项目研究获得结果可为精子生成障碍提供分子信息，并为精子成熟异常导致的不育症诊治提供分子靶。

piRNA是一类动物生殖细胞特异性小分子非编码RNA。研究发现,piRNA特异性地与PIWI蛋白结合，沉默基因组移动遗传元件，维持生殖细胞基因组稳定性和完整性，为配子形成所必需，但目前对于PIWI/piRNA在生殖细胞中是否还发挥沉默转座元件以外的其它功能还了解不多。在该项目支持下，我们研究了小鼠PIWI蛋白（MIWI）及piRNA在小鼠精子细胞发育及精子形成中的新功能，并探索了HIWI（人PIWI）蛋白突变在无精症发生中的功能机制，主要获得了以下研究结果，包括：1）发现小鼠piRNA在精子形成后期触发其结合蛋白PIWI（MIWI）经APC/C-泛素化降解的新途径和piRNA诱导PIWI降解的新功能，揭示piRNA与MIWI蛋白以协同模式在精子形成后期被共同清除、piRNA作为“配体”调控其结合蛋白泛素化修饰的新机制，并提供了一种新型的蛋白泛素化降解调控模式；2）从无精患者中鉴定了拮抗HIWI（人源PIWI）蛋白泛素化修饰的D-box突变，并通过Knock-in小鼠模型发现，此类突变阻碍精子变形过程中的组蛋白-鱼精蛋白交换并导致精子形成异常及雄性不育；3）发现小鼠粗线期piRNA与其结合蛋白MIWI和脱腺苷酶CAF1组成pi-RISC复合物，指导精子细胞中mRNA大规模地脱腺苷酸化及降解，提供了精子发生后期父本mRNA清除的一种重要分子机制，揭示了piRNA除了沉默转座元件以外，还具有调控生殖细胞编码基因表达的新功能。总之，我们的这些发现揭示了PIWI/piRNA在哺乳动物精子形成过程中的一系列新功能。.发表资助项目标注6篇，包括研究论文5篇[Cell, accepted; Cancer Res, 2017, 77(1):100-111; Cell Res, 2014, 24(2):254-257；Cell Res, 2014, 24(6):680-700; Dev Cell, 2013, 24(1): 13-25]、应邀综述论文1篇[WIREs RNA, 2014, 5(6):733-745]，申请发明专利一项（申请号201610525017）。此外，项目负责人在项目执行期间获得基金委“国家杰出青年科学基金”，培养了5名博士研究生毕业取得博士学位，4名硕士研究生毕业取得硕士学位。